This is a proposal to renew a Research Resource grant that supports the Boulder Laboratory for 3-D Electron Microscopy of Cells. Our goals for research in technology include the development of methods for reliable specimen preparation, image acquisition, and image processing. The methods we envision should ultimately reveal the 3-D structure of large volumes from cells and organelles at space resolutions that have previously been unattainable. This goal will be achieved through the study of vitrified cells, an approach that preserves both large-scale cellular organization and the atomic details of macromolecular complexes within cells. We will work to visualize details at 2 - 3 nm, a resolution sufficient to dock atomic models of macromolecules into 3-D cryo-EM images, by using cryo-electron tomography of various thin, frozen hydrated samples. These will including very thin cells, isolated organelles and macromolecular complexes, and sections prepared by cryo-microtomy. Our studies of large-scale features will use vitrified samples that are subsequently fixed by freeze-substitution and embedded in plastic for microtomy, allowing the examination of big fractions of any given mammalian cell, either in culture or within the context of its normal tissue. These approaches will be integrated through image processing to generate a comprehensive view of cellular structure and function. By connecting these imaging modalities, we should be able to interpret data from light microscopy and physiological studies of living cells in terms of the molecular details that can be observed through the combination of high-resolution cryo-tomography with atomic resolution structures. Our technological studies are motivated in part by our collaborations with seven labs whose biomedical research has posed scientific questions that require the kind of high resolution and/or large volume 3-D information about cells that our methods should provide. Most of these collaborations and many of our service projects are directly related to human diseases and disorders, so our technological work should soon inform medical science. Some of the structures we will study are currently under investigation as targets for drugs and other therapeutic measures, so our detailed cellular and molecular structure studies should also help translational research. Our proposal also describes plans for training and dissemination, so the use of our technologies will soon become widespread. [unreadable] [unreadable] [unreadable] [unreadable]